SH-SY5Y Cell Culture

The human neuroblastoma SH-SY5Y cells were cultured in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin G (100 U/mL) and streptomycin (0.1 mg/mL). Cells were cultured at 37°C in a 5% CO₂ atmosphere. The SH-SY5Y cells were seeded at the density of 3000 cells/well in 96-well microplate for the MTT assay, measurement of mitochondrial membrane potential, intracellular calcium levels, and reactive oxygen species (ROS) assays. The cells were incubated with 6-OHDA and different concentration of morphine for 24 h. Morphine was added 24 h before 6-OHDA. The ER stress inhibitor 4-phenylbutyrate (4-PBA) and autophagy inhibitor 3-Methyladenine (3-MA) were added 1 h before incubation of morphine.